酶催化中静电作用的核磁共振研究

The catalytic effect of enzyme should be primarily due to the electrostatic effects. However, because of the complex composition and structure of protein (including the solvent effect), the electrostatic interaction displays a strong heterogeneity, and the quantitative measurement is always an enormous challenge. Therefore, either the mechanism of electrostatic interaction in protein or the relationship between electrostatic interaction and enzymatic activity is still not clear. In previous work, we have developed a new method based on the nuclear magnetic resonance (NMR) technique to measure the electrostatic interaction in protein simply and accurately. It provides a new opportunity to further clarify the mechanism of electrostatic interaction and its influence on enzyme catalysis. In this project, combining the new NMR technique with computer simulation, we attempt to explore the mechanism of the electrostatic interaction heterogeneity and the source of electrostatic contribution to enzymatic activity. The highly stable staphylococcal nuclease (SNase), which catalyzes the hydrolysis of both DNA and RNA at the 5' position of the phosphodiester bond, is selected as the study system. The successful implementation of this project will improve the understanding of the electrostatic interaction mechanism in protein and the relationship between electrostatic interaction and enzymatic activity. It would provide new thoughts for function regulation of enzymes.

静电作用被认为是稳定酶催化反应过渡态的主要因素之一。然而，由于测量方法的局限，目前蛋白质静电作用微观机制的实验解析依然困难，静电作用影响酶活性的微观机制尚不清晰。前期工作中，我们发展了基于核磁共振的静电测量方法，可以显著提高静电作用力的测量精度。以此为契机，本项目拟结合核磁共振和计算模拟，以核酸裂解酶SNase为研究对象，定量研究其不同区域的静电作用，在探索不同区域介电常数异质性机制的基础上解析不同区域、不同方向静电作用力对反应过渡态结构和能垒的影响，阐明静电作用对酶活性的影响机制。本项目的实施将促进对蛋白质静电作用机制以及静电作用与酶催化活性关系的理解，为酶功能调控探索新思路。

由于测量方法的局限，静电作用影响酶活性的微观机制尚不清晰。前期工作中，我们发展了基于核磁共振的静电测量方法，显著提高了静电作用力的测量精度。以此为契机，本项目结合核磁共振、酶学实验和计算模拟等，以木聚糖水解酶BCX为研究对象，解析了静电作用扰动体系熵影响酶催化活性的微观机制。我们发现在BCX催化过程中存在对催化有利的熵增效应，而远程静电作用对催化位点的扰动也主要是通过熵增效应实现的。因此我们推测静电作用-熵-催化活性相互关联。为了验证此推测，我们首先通过QM/MM计算获得了催化限速步的过渡态结构和电荷分布，然后基于基态结构执行多温度副本交换模拟解析由基态电荷变为过渡态电荷导致的熵变化，并拆分到活性中心的各个残基上，发现N35、Y65和R122是熵增效应的主要来源；随后，构建了N35A、Y65A和R122A突变体加以实验验证，发现N35和Y65变为A后熵增现象消失，且活性均有所降低，R122A活性更是弱到难以检测，证明从基态到过渡态电荷变化过程中N35、Y65和R122残基相关的作用力和动态性变化是BCX催化中熵增效应的重要来源。为了进一步验证静电作用扰动是熵效应主要来源的结论，我们又做了不同盐浓度的对比实验，发现随着盐浓度升高，静电屏蔽效应变强，催化过程中的熵增效应逐渐变弱直至变为熵减，与预期相符。此工作在实验上真正关联了静电作用-熵-催化活性，为理解酶催化机制和改进酶功能提供了新途径和新思路。此外，受上述工作启发，我们还开展了多项与蛋白质结构动态性（熵）相关的研究：发展了一种精度和分辨率更高的蛋白质动态性检测方法；揭示了结构动态性变化改变CohA2结合模式的现象和机制；采用增强动态性使熵增的策略将前药酶yCD的活性提高至野生型的3倍左右；从动态性（熵）角度解析了CBM蛋白S-糖基化和O-糖基化差异导致的底物选择性差异等。